Hi fbs

We isolated PBMCs from healthy donors and cryopreserved these cells to titrate and validate our antibody panel as well as to use in our single control and FMO compensation assays. Approximately 250 mL of healthy donor whole blood in EDTA tubes was acquired from Research Blood Products LLC Boston, delivered at RT. Upon delivery, whole blood was diluted 1:1 in 2% heat-inactivated fetal bovine serum (Hi-FBS) (Sigma, Saint-Louis, MO, USA, cat#F4135) in Hanks’ Balanced Salt Solution (HBSS), without calcium or magnesium (Thermo Fisher, Waltham, MA, USA, cat#14170112), i.e., washing buffer (WB). After careful mixing, the suspension was layered on top of an equal volume of Lymphoprep density gradient medium (Stem Cell, Vancouver, BC, Canada, cat#07811) in a 50-mL tube and centrifuged at 800× g for 20′ at RT with brakes off. Afterwards, using a sterile dropper pipet, the PBMC layer was collected in a new 50-mL tube and washed with WB. PBMCs were next centrifuged at 400× g for 10′ at RT with brakes on. The tube was decanted, flicked 3 times, and the PBMC pellet was resuspended in 50 mL WB. The tube was again centrifuged at 400× g for 10′ at RT, then resuspended in WB and counted before subsequent cryopreservation.

The cell lines were obtained from ATTC (Manassas, VA, USA). HeLa (human cervix epithelioid carcinoma), MCF7 (human breast adenocarcinoma), HepG2 (human hepatocellular carcinoma), U87 MG (human glioblastoma), T98G (human glioblastoma), GL261 (murine glioma), LNCaP (human prostate cancer), PC-3 (human prostate cancer), and HEK-293 (human embryonic kidney) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Waltham, MA, USA), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (HIFBS, Sigma-Aldrich, St. Louis, MO, USA)), 2 mM L-glutamine, 100 μg/mL streptomycin, and 100 units/mL penicillin (Gibco, Waltham, MA, USA) at 37 °C in saturated humidity atmosphere containing 95% air and 5% CO₂. Cell line HT-29 (human colon carcinoma) was grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Whaltman, MA, USA) supplemented with 10% HIFBS, 100 μg/mL streptomycin, and 100 units/mL penicillin (Gibco, Whaltman, MA, USA) at 37 °C in humidified 95% air and 5% CO₂ atmosphere. MycoAlert kit (Lonza, Norwest, Australia) was used to routinely check the presence of mycoplasma, and only mycoplasma-free cells were employed in the experiments.

HepG2 were purchased from ATCC and HuH7 cells were a kind gift from Dr. Peter Sorger (Harvard Medical School). Hepatocytes used for P. berghei infections were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with L-glutamine (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) (v/v) (Sigma-Aldrich) and 1% antibiotic-antimycotic (Thermo Fisher Scientific) in a standard tissue culture incubator (37°C, 5% CO₂). Anopheles stephensi mosquitoes infected with luciferase- or GFP-expressing P. berghei ANKA were purchased from the NYU Langone Medical Center Insectary Core Facility. Sporozoites used for liver stage experiments were harvested from freshly dissected salivary glands of mosquitoes.
P. falciparum parasites of the 3D7 strain (ATCC) were maintained in red blood cells (Golf Coast Regional Blood Center) cultured in RPMI 1640 medium supplemented with 0.5% (m/v) AlbuMAX II, 25 mM HEPES, 25 ug/mL gentamycin, 24 mM sodium bicarbonate and 50 μg/mL hypoxanthine at a pH of 7.2 and maintained at 37°C under 92% N₂, 5% CO₂, and 3% O₂. Synchronization was performed using 5% sorbitol as previously described [58 (link)].

Each of the 24 tissue sections for cryopreservation was placed in a cryovial containing 1mL chilled cryopreservation media (10% DMSO, 90% HI-FBS, both Sigma). Cryovials were cooled to -80°C at 1°C /min using a freezing container (Mr. Frosty, Nalgene), then transferred to the vapor phase of liquid nitrogen for 2–5 days. Following cryopreservation, cryovials were removed from liquid nitrogen and agitated gently in a 37°C water bath until just thawed. Thawed tissues were transferred to 25mL of R10 for 10 minutes to allow DMSO to elute from the tissue.

Promastigotes of L. amazonensis (strain MHOM/BR/75/Josefa, 5 x 10⁵/mL), L. braziliensis (strain MHOM/BR/75/M2903, 2 x 10⁶/mL) and L. infantum (strain MHOM/MA67ITMAP263, 2 x 10⁶/mL) at the logarithmic phase of growth, were incubated at 26°C with different concentrations of compounds 1c, 2 and 3 for 72 h in M199 medium containing 5% of heat inactivated fetal bovine serum (HIFBS), 0.2% hemin (Sigma-Aldrich), 100 μg/mL streptomycin and 100 UI/mL penicillin (Sigma-Aldrich). Parasite viability was assessed in the last four hours of culture by Alamar blue (ThermoFisher Scientific). The concentrations that reduced cell viability by 50% (EC₅₀) were calculated by non-linear regression analysis using GraphPad Prism 7 software.
For mutant cell lines, promastigotes of L. mexicana T7 Cas9 (Parental), Δ1040, Δ1160 and ΔcTXNPx (2 x 10⁶/mL), were incubated at 26°C with different concentrations of compounds 1c for 48 h in Schneider’s medium (Sigma-Aldrich) containing 10% of heat inactivated fetal bovine serum (HIFBS), 100 μg/mL streptomycin and 100 UI/mL penicillin. Parasite viability and EC₅₀ values were assessed as described above.

Monocytoid THP-1 cells were obtained from ATCC (Manassas, VA, USA) and were cultured in RPMI-1640 medium, supplemented with 10% heat-inactivated foetal bovine serum (HIFBS), 2 mM l-glutamine, 100 U/100 µg/ml penicillin/streptomycin and 0.05 mM 2-mercaptoethanol (all from Sigma, St Louis, MO, USA). For differentiation and polarisation of THP-1 cells to macrophages, 25 ng/mL PMA, 20 ng/mL IFNγ, 100 ng/mL LPS, 20 ng/mL IL4 and 20 ng/mL IL13 (final concentrations) were used according to an established protocol with minor modifications^{14 (link)}. The SRE Reporter HEK293 cell line was obtained from BPS Bioscience (San Diego, CA, USA) and used for monitoring the activity of the MAPK/ERK signalling pathway. The SRE Reporter HEK293 cell line contains a firefly luciferase gene under the control of Serum Response Element (SRE) responsive elements stably integrated into HEK293 cells, resulting in an ERK pathway-responsive reporter cell line. All cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO2. CD4+ T-cells from patients with SLE were purchased from Astarte Biologics, LLC (Bothell, WA, USA) and handled according to the manufacturer’s recommendations.